1. Field of the Invention
The present invention relates to a method for isolation and cultivation of an inner cell mass, and a method for preparation of embryonic stem cell lines using the inner cell mass isolated by the same.
2. Description of the Related Art
A sperm is combined with an ovum to form a single fertilized ovum and the fertilized ovum begins cell division and forms a solid sphere of cells called a blastocyst. The blastocyst possesses an inner cell mass (ICM), which comprises plural cells to form an embryo through cell division and cell differentiation. The embryo develops into a fetus, then, an individual after a gestational period.
When cells of the inner cell mass are isolated from the blastocyst and cultured under desired conditions, a cell having potential for differentiation may be obtained although no more differentiation proceeds in this cell. Such cell refers to an embryonic stem cell. Briefly, the embryonic stem cell is a cell derived from the inner cell mass of an early stage of embryo, and the embryonic stem cell remains undifferentiated but has potential to differentiate into any type of cell and/or tissue found in organs of an individual.
In other words, the embryonic stem cell is pluripotent. The embryonic stem cell may indefinitely repeat cell division in theory. Based on these characteristics, it is expected the embryonic stem cell may be effectively used for regeneration of a tissue after injury or disease. In particular, a number of researches and investigation into embryonic stem cell therapies have been increasingly conducted since Thomson et. al. (1998) succeeded in cultivation of human embryonic stem cells.
Accordingly, isolation of an inner cell mass as a precondition for formation of the embryonic stem cell has also been recently studied. Among such isolation methods, immunosurgery is well known and commonly used. The immunosurgery method will be described as follows.
First, a zona pellucida of a fertilized ovum is dissolved in 0.1% pronase for 1 to 2 minutes. Then, a blastocyst cell mass present in the zona pellucida is stored in a 100% anti-human serum antibody solution (Sigma) for about 20 minutes, followed by storing again the blastocyst cell mass in a guinea pig complement for about 30 minutes, so as to destroy a trophoblast layer and isolate the inner cell mass from the blastocyst. As described above, such immunosurgery method for isolation of an inner cell mass is based on chemical treatment.
The isolated inner cell mass by immunosurgery is fixed to a fetal fibroblast (that is, feeder cell) then proliferated and, when a density and a size of the proliferated cell mass are increased, it may be considered that formation of an embryonic stem cell line is completed. Such formed embryonic stem cell is separated into smaller cell colonies, which are in turn transferred to a fresh culture dish (such as Petri-dish) under the same culturing conditions in order to repeat subculturing thereof, so that the cell colonies are continuously proliferated while remaining undifferentiated. Repeating the above processes may produce a number of embryonic stem cells.
However, since the immunosurgery described above adopts immunological and/or chemical treatment to destroy a blastocyst in order to isolate an inner cell mass contained in the blastocyst, the isolated cells often fail to be fixed to the feeder cell. Further, such chemical treatment encounters a problem in that the isolated inner cell mass cannot remain un-differentiated, instead being differentiated into undesired cells, therefore, the desired un-differentiated embryonic stem cell line may not be efficiently established.
Accordingly, the immunosurgery method has a restriction in number of embryonic stem cell lines successfully established by culturing plural cells on a feeder cell, since the number of the embryonic stem cell lines generally ranges from 10 to 20% of a total number of fertilized ova used in the isolation.